Spontaneous calcium oscillations regulate human cardiac progenitor cell growth

Abstract

Rationale: The adult heart possesses a pool of progenitor cells stored in myocardial niches, but the mechanisms involved in the activation of this cell compartment are currently unknown.

Objective: Ca2+ promotes cell growth raising the possibility that changes in intracellular Ca2+ initiate division of c-kit-positive human cardiac progenitor cells (hCPCs) and determine their fate.

Methods and results: Ca2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca2+. These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca2+ oscillations in hCPCs were regulated by the release of Ca2+ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca2+ by the sarco-/endoplasmic reticulum Ca2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na+-Ca2+ exchanger, store-operated Ca2+ channels and plasma membrane Ca2+ pump were present and functional in hCPCs, they had no direct effects on Ca2+ oscillations. Conversely, Ca2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca2+ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny.

Conclusion: IP3R-mediated Ca2+ mobilization control hCPC growth and their regenerative potential.

Figure 1

Intracellular Ca²⁺ in hCPCs. A, Cytosolic Ca²⁺ levels in a quiescent hCPC (upper trace) and in two hCPCs showing single (middle trace) and multiple (lower trace) Ca²⁺ oscillations. B, Distribution of Ca²⁺ oscillations, from 1 to more than 8, in hCPCs over a period of 33 min. C, Amplitude and duration of Ca²⁺ events in hCPCs. D, Ca²⁺ oscillations in hCPCs (Active Cells) analyzed for a period of 132 minutes. E, Ca²⁺ oscillations in hCPCs in control condition (Ctrl) and at the G₁-S and G₂-M transition.

Figure 2

Cell-to-cell interaction and Ca²⁺ oscillations. A-C, Cascade blue (blue) microinjected in a single hCPC (A) translocated spontaneously to adjacent cells (B and C). D, Rhodamine-labeled dextran (red), delivered simultaneously with cascade blue, remained confined to the injected cell. Scale bar: 20 μm. Cascade blue translocation was detected in 6 experiments. E, Connexin 43 (Cx43, white) is present between hCPCs (c-kit, green). Nuclei are stained by DAPI (blue). Scale bar: 20 μm. A group of cells is shown at higher magnification on the right panel. The inset shows Cx43 labeling in a myocyte [α-sarcomeric actin, (α-SA) red]. Scale bar: 10 μm. F, Intracellular Ca²⁺ in hCPCs before (left trace) and after exposure to octanol (right trace). G, Effects of uncoupling on Ca²⁺ oscillations.

Figure 3

Ca²⁺ cycling in myocytes and CPCs. A, Cx43 (white) between DiI-labeled hCPCs (DiI, red) and myocytes (α-SA, green). Nuclei are identified by DAPI. Scale bars: 10 μm. B, Intracellular Ca²⁺ in hCPCs (blue traces) and adjacent co-cultured neonatal myocytes (black traces). The effects of electrical stimulation and cadmium chloride are also shown. Different cells were used in the three conditions. C, Intracellular Ca²⁺ in hCPCs and neonatal myocytes in line scan mode. Red identifies hCPCs loaded with DiI and green corresponds to Fluo-3; scale bar: 20 μm. D, Cytosolic Ca²⁺ in a quiescent mCPC (upper trace) and two mCPCs showing a single (middle trace) and multiple (lower trace) Ca²⁺ oscillations. E, Properties of Ca²⁺ oscillations in hCPCs and mCPCs. E, c-kit-EGFP mouse heart loaded with Rhod-2 (red), stimulated at 1 Hz and analyzed in line-scan mode (arrows). CPCs were identified by EGFP (green). G, Ca²⁺ transients in myocytes (red trace) did not affect Ca²⁺ levels in the neighboring EGFP-positive CPC (green trace). Identical results were obtained in 7 other experiments. Scale bars: 20 μm.

Figure 4

Ca²⁺ regulatory proteins in hCPCs. A-D, Expression at the mRNA (A) and protein (B-D) levels of the components of the ER that are implicated in Ca²⁺ homeostasis. Myocytes were used as positive control for RyRs. Human heart (hHeart) was used as positive control. Scale bars: 20 μm. E, Repetitive Ca²⁺ oscillations in hCPCs in the presence of IP3R agonist. F-H, Ca²⁺ oscillations in hCPCs at baseline (Tyrode) and in the presence of activation (F) and inhibition (G and H) of IP3R function. Xestopsongin-C, XeC. I-N, Ca²⁺ in hCPCs in the presence of modulators of SERCA (I and J) and RyRs (K-N). *P<0.05 vs. Tyrode.

Figure 5

G_q-protein coupled receptors and intracellular Ca²⁺ in hCPCs. A-C, Expression at the mRNA (A) and protein (B and C) levels of P2Y2 and H1 receptors in hCPCs. Human heart (hHeart) was used as positive control. Scale bars: 10 μm. D-G, Ca²⁺ oscillations in hCPCs in the presence of ATP (D and E) or histamine (F and G). *P<0.05 vs. Tyrode.

Figure 6

hCPC growth and apoptosis. A-C, ATP and histamine increase Ca²⁺ oscillations and proliferation of hCPCs. Inhibitors of Ca²⁺ oscillations prevent the effects of ATP and histamine. Control, Ctrl; histamine, His. *P<0.05 vs. Ctrl, **P<0.05 vs. agonist. D and E, Apoptosis of hCPCs measured by Annexin V labeling and FACS. PI, propidium iodide. Q2, late apoptotic or necrotic cells; Q3, alive cells; Q4, cells undergoing apoptosis. F and G, IGF-1R transcript and protein in hCPCs and hHeart. Scale bar: 20 μm. Right panel in G illustrates selected cells at higher magnification. H and I, Intracellular Ca²⁺ in hCPCs exposed to IGF-1. *P<0.05 vs. Tyrode. J, Proliferation of hCPCs in the presence of IGF-1 alone or in combination with inhibitors of Ca²⁺ oscillations. *P<0.05 vs. Ctrl, ** P<0.05 vs. IGF-1.

Figure 7

Ca²⁺ oscillations and growth of hCPCs in vivo. A, EGFP-positive hCPCs 48 hours after implantation in the infarcted mouse heart under control conditions and following activation of hCPCs with ATP or histamine. Proliferation of EGFP-positive cells (green) is documented by BrdU labeling (magenta, arrows). B, Nkx2.5 (white) is present in several EGFP-positive cells (arrows). Scale bars: 20 μm. C, Results are shown as mean±SEM. *P<0.05 vs. Ctrl.

Figure 8

Myocardial regeneration by activated hCPC. A, Mouse heart treated with histamine-stimulated hCPCs. The mid-portion of the infarct is replaced by EGFP-positive (upper panel, green) α-SA-positive cardiomyocytes (lower panel, red). The area in the rectangle is shown at higher magnification in the inset. B, Extent of regeneration mediated by hCPCs non activated (Ctrl hCPCs) or exposed to ATP or histamine (ATP-His hCPCs). C, LV function in sham operated (SO), infarcted untreated (MI + PBS) and hCPC-treated (MI + hSCPs) mice 7 days after coronary ligation. Ctrl, ATP and His identify non-stimulated, ATP-stimulated and histamine-stimulated hCPCs, respectively. LVEDP, LV end-diastolic pressure; LVDevP and +dP/dt. *P<0.05 vs. SO, **P<0.05 vs. MI + PBS, † P<0.05 vs. MI injected with untreated hCPCs.